传染性法氏囊病病毒株VP2基因在重组腺病毒中的表达  

Expression of VP2 gene of infectious bursal disease virus from immunoprophylaxis defeat's chickens in recombinant adenovirus

作　　者：潘群兴[1] 王永山[1] 何孔旺[1] 欧阳伟[1,2] 王晓丽[1] 王忠灿[3] 唐雨德[3] 

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095 [3]南京军区疾病预防控制中心,江苏南京210002

出　　处：《中国兽医学报》2010年第3期312-316,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30571371);江苏省自然科学基金资助项目(BK2008065;BK2008352);江苏省农业科技创新项目(CX(08)106)

摘　　要：用RT-PCR方法从传染性法氏囊病(IBD)免疫预防失败的病鸡法氏囊组织AH1与AH2中扩增传染性法氏囊病病毒(IBDV)VP2基因。序列分析结果显示,AH1与AH2病毒VP2基因长度均为1350nt,编码450aa,核苷酸和氨基酸序列的同源性分别为98.2%、99.3%,七肽基序均为SWSASGS,在222、253、256、279、284、294和299位上的氨基酸残基分别是A、Q、I、D、A、I和S,具有IBDV强毒的分子特征。进一步将VP2基因克隆入人5型腺病毒穿梭载体(pShuttle-CMV),与腺病毒骨架载体(pAdEasyTM)共转化大肠杆菌BJ5183进行同源重组并转染HEK-293A细胞,经多次亚克隆获得了重组腺病毒rAd-(IBDV)VP2。利用Western-blot、IFA等方法检测IBDVVP2蛋白的体外表达情况,结果证明VP2基因在腺病毒中获得了表达。VP2 genes of infectious bursal disease virus （IBDV） were amplified by reverse transcription-polymerase chain reaction （RT-PCR） and cloned from bursae of Fabricius AH1 and AH2 collected from 2 chicken farms of Anhui province of China in December 2007,which immunized with infectious bursal disease （IBD） vaccine but attacked by IBDV at 2 weeks post the immunization.Sequence analysis showed that the VP2 gene of the current local IBDV isolates AH1 and AH2 has 1 350 nucleotides and encode 450 amino acids.The homology of the nucleotide and amino acid sequences are 98.2% and 99.3%,respectively.Both have the same heptapepetide motif （S-W-S-A-S-G-S） and critical amino acid residues A222,Q253,I256,D279,A284,I294 and S299 in the variable region of VP2,suggesting that AH1 and AH2 possess the molecular characteristics of virulent IBDV.Furthermore,the VP2 gene was cloned into the transfer vector pShuttle-CMV（named pShuttle-VP2）.The pShuttle-VP2 and bone vector pAdEasy-1 were cotransformed into BJ5183 to generate the recombinant plasmids pAd-Cap with homologous recombination.The recombinant adenovirus rAd-（IBDV）VP2 was generated by transfection and plaque purification in HEK-293A cells.Expression of VP2 proteinin infected 293 cells was confirmed by Western-blot and IFA.

关 键 词：传染性法氏囊病病毒 VP2 重组腺病毒 表达 

分 类 号：S852.65[农业科学—基础兽医学]